1097-pET28b-T4-G32
Cloning of 1097-pET28b-T4-G32
[edit]Purpose: Cloning of 1097-pET28b-T4-G32
Date: 2012-10-5---2012-10-9
Notebook:0
Page:
Clone ID:1097
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR using the following primers and T4 phage DNA,
T4_G32-Nde1-F+T4_G32-Xho1-R
clone into Nde1 + Xho1 digested 1027 pET28b-LambdaBet
Colony Screening
[edit]Date: 2012-10-9
no clonies in plate
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]&
Cloning of 1097-pET28b-T4-G32
[edit]Purpose: Cloning of 1097-pET28b-T4-G32
Date: 2012-10-12---2012-10-15
Notebook:0
Page:
Clone ID:1097
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR using the following primers and T4 phage DNA,
T4_G32-Nde1-F+T4_G32-Xho1-R
| lane 6 |
lane7 | |
| PCR product |
1026-pET28b-LambdaExo | |
| enzymes |
Nde1+Xho1 | |
Colony Screening
[edit]Date: 2012-10-15
mini prep and digest with Nde1+Xho1
clone 1,2 need to sequence,
sequence result shows clone1is not the right clone, 1026 contamination
Result intepretation/Summary
[edit]Conclusion
[edit]Cloning of 1097-pET28b-T4-G32
[edit]Purpose: Cloning of 1097-pET28b-T4-G32
Date: 2012-12-26
Notebook:0
Page:
Clone ID:1097
Experimenters: Jennifer Truong
Results
[edit]cloning
[edit]
See lane 1
PCR using the following primers and T4 phage DNA,
clone into Nde1 + Xho1 digested 1027 pET28b-LambdaBet
Cut out lane 3 (insert) and 4 (vector) and ligate together overnight
Date: Jan 3, 2012
See lane 1 and 4
USING THE FOLLOWING AMOUNT OF TEMPLATE
Lane 1: 1:10 dilution of straight Phage DNA, 1 uL
Lane 2: 1:10 dilution|} of straight Phage DNA, 5 uL
Lane 3: 1:10 dilution of straight Phage DNA, 10 uL
Lane 4: 5 uL of purified Phage DNA
Lane 5: 1 uL of unpurifed Phage DNA
Lane 6: 3 uL of unpurified Phage DNA
PCR using the following primers and T4 phage DNA
T4_G32-Nde1-F
acgata CATATG atgtttaaacgtaaatctactgc
T4_G32-Xho1-R
(gatgaccttttgaatgacctttaaCTCGAG cgagca)
tgctcgCTCGAGttaaaggtcattcaaaaggtcatc
Clone into Nde1 + Xho1 digested 1279 pT7A3
Colony Screening
[edit]Result intepretation/Summary
[edit]
Conclusion
[edit]
Cloning of 1097 pT7A3-T4-G32
[edit]Purpose: Cloning of 1097 pT7A3-T4-G322
Date: 2013-1-5--
Notebook:0
Page:
Clone ID:1097
Experimenters: shanyi
Results
[edit]cloning
[edit]PCR
digest with Nde1 + Xho1(red is right)
[Image:1049 1097 1123DIGEST-right.JPG]]
Colony Screening
[edit]
Protein Expression
[edit]2013/3/29
SHANYI
| Lane |
Name |
Protein Name |
Clone ID |
Host |
Induction |
Purification |
pI/Mw |
| M |
PageRuler Plus Prestained protein ladder |
|
|||||
| 1 |
pT7A3-T4-G32 |
T4-G32 |
1097 |
5 ml, Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
4.82 / 33506.43 |
| 2 |
pT7A3-MBP-(3c)-MutM |
MBP-(3c)-MutM |
1138 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
8.68 / 30290.07+43.6 |
| 3 |
pT7A3-KOD |
KOD |
1458 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
7.53 / 90022.42 |
| 4 |
pT7A3-UDG |
UDG |
1494 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
5.45 / 27174.73 |
| 5 |
pT7A3-T7-Endo1 |
T7-Endo1 |
1505 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
9.54 / 17171.84 |
| 6 |
pT7A3-polyAPol |
polyAPol |
1513 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
9.67 / 53870.91 |
| 7 |
pT7A3-T7-Exo |
T7-Exo |
1558 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
4.93 / 34502.20 |
| 8 |
pT7A3-MBP-(3C)-Exo-1 |
MBP-(3C)-Exo-1 |
1562 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
8.80 / 18143.72 |
| 9 |
pT7A3-MBP-(3C)-RnaseH |
MBP-(3C)-RnaseH |
1573 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
8.43 / 17596.99 +43.6 |
| 10 |
pT7A3-MBP-(3c)-E_coli_Endo IV |
MBP-(3c)-E_coli_Endo IV |
1593 |
5 ml,
Nico21(DE3)plysN |
0.2mM IPTG RT o/n |
Ni beads |
5.43 / 31479.53 +43.6 |
| 11 |
pT7A3-T3 DNA ligase |
T3 DNA ligase |
1654 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
5.14 / 39350.70 |